Antibiotics have proven to be one of medicine's most effective tools in combating disease, but their utility is constantly being challenged by the emergence of antibiotic-resistant target organisms and their future effectiveness is now in doubt. The pharmaceutical industry responds to a newly-emerged resistant strain by developing a modified version of the drug that was effective against a predecessor of the newly-emerged resistant strain, and in turn pathogens evolve resistance to the new drug. Some of the best evidence that drug resistance genes can evolve by mutation to confer resistance to newer drugs comes from the TEM/SHV β-lactamases in which genes that originally did not confer resistance to 2nd and 3rd generation cephalosporins evolved the ability to do so as the result of base substitution mutations. The products of those evolved genes are called extended-spectrum β-lactamases (ESBL's). The example of ESBL's has led to a paradigm according to which the acquisition of resistance to new drugs will almost always involve evolution of known drug resistance genes as the result of selectively advantageous mutations.
This paradigm ignores the issue of the ultimate source of antibiotic resistance genes. Understanding the origins of antibiotic resistance genes may be an essential key to predicting how resistance to new drugs will arise.
In most cases, the source of resistance genes is unknown. Most resistance genes were originally detected on plasmids and, although they spread from plasmid-to-plasmid and via plasmids from one bacterium to another, their original source cannot readily be determined. The ampC β-lactamase genes, whose products are the Class C β-lactamases, provide a rare exception to the ignorance of resistance gene origins.
Class C β-lactamases are generally quite active toward cephalosporins, including the third generation derivatives, but have not been taken as a serious threat until recently, because the genes for Class C β-lactamases are typically located on chromosomes rather than on plasmids. The chromosomal ampC genes are found in a variety of Gram negative bacteria, including both the Enterobacteriaceae and Pseudomonas species. ampC genes are typically expressed at a low level, as in E. coli, or are inducible by penicillins and early generation cephalosporins but not inducible by third and fourth generation cephalosporins. Over the last decade, however, it has been found that Gram negative pathogens that are hyper-producers of ampC β-lactamases are resistant to all but a few of the most recently introduced β-lactam antibiotics (Livermore, “Are all Beta-lactams Created Equal?” Scand. J. Infect. Dis. Suppl. 101:33–43 (1996)). By now 25–50% of Enterobacter isolates from intensive care unit patients in many major Western and Far Eastern hospitals are AmpC hyper-producers and are resistant to all penicillins and cephalosporins except imipenem, meropenem and temocillin (Livermore, “Are all Beta-lactams Created Equal?” Scand. J. Infect. Dis. Suppl. 101:33–43 (1996)). Even more worrying are several recent reports of de-repressed ampC genes located on plasmids found in pathogenic Gram negative bacteria (Bauernfeind et al., “Characterization of the Plasmidic Beta-lactamase CMY-2, which is Responsible for Cephamycin Resistance,” Antimicrob. Agents Chemother. 40:221–224 (1996); Horii et al., “Characterization of a Plasmid-borne and Constitutively Expressed blaMOX-1 Gene Encoding AmpC-type Beta-lactamase,” Gene 139:93–98 (1994); Jacoby and Medeiros, “More Extended-spectrum Beta-lactamases,” Antimicrob. Agents Chemother. 35:1697–1704 (1991); Papanicolaou et al., “Novel Plasmid-mediated Beta-lactamase (MIR-1) Conferring Resistance to Oxyimino- and Alpha-methoxy Beta-lactams in Clinical Isolates of Klebsiella pneumoniae,” Antimicrob. Agents Chemother. 34:2200–2209 (1990)). Although Amyes (“Genes and Spectrum: The Theoretical Limits,” Clin. Infect. Dis. 27 Suppl 1:S21–28) suggests that AmpC β-lactamases presently do not seem efficient enough to cause widespread clinical problems, others are quite concerned that the AmpC β-lactamases constitute a pool from which clinically significant resistant strains may well emerge (see Lindberg and Normark, “Contribution of Chromosomal Beta-lactamases to Beta-lactam Resistance in Enterobacteria,” Rev. Infect. Dis. 8 Suppl 3:S292–304 (1986); Morosini et al., “An Extended-spectrum AmpC-type Beta-lactamase Obtained by in vitro Antibiotic Selection,” FEMS Microbiol. Lett. 165:85–90 (1998); Pitout et al., “Antimicrobial Resistance with Focus on Beta-lactam Resistance in Gram-negative Bacilli,” Am. J. Med. 103:51–59 (1997)). That concern is exacerbated by the finding that in a clinical isolate of Enterobacter cloacae, the AmpC β-lactamase has extended its substrate range to include the oxyimino β-lactams as the result of a tandem duplication of three amino acids (Nukaga et al., “Molecular Evolution of a Class C Beta-lactamase Extending its Substrate Specificity,” J. Biol. Chem. 270:5729–5735 (1995)(erratum published at J. Biol. Chem. 270(36):21428)). More recently, Morosini and colleagues (“An Extended-spectrum AmpC-type Beta-lactamase Obtained by in vitro Antibiotic Selection,” FEMS Microbiol. Lett. 165:85–90 (1998)) applied direct selection to isolate a mutant of an Enterobacter cloacae AmpC β-lactamase in which the activity toward a 4th generation cephalosporin had increased 267-fold as the result of a single amino acid replacement.
Together, the facts that mutations to hyper-expression of AmpC occur readily and hyper-expressed ampC β-lactamases (a) already confer resistance to penicillins and 3rd generation cephalosporins, (b) are moving onto plasmids, and (c) can easily mutate to significant activity against 4th generation cephalosporins suggest that AmpC β-lactamases may very well constitute a potentially serious clinical threat.
The above observations make it clear that new antibiotic resistance can arise when more or less primitive, chromosomal, antibiotic genes are mobilized onto plasmids. Thus, a need exists to identify putative antibiotic resistance genes before they become effective and can cause great damage to the public health. With the genome sequences of literally hundreds of microorganisms now being determined, opportunities are now available to identify those suspected resistance genes, to determine whether they can, in fact, become clinical antibiotic resistance determinants, and to design drugs that can remain effective in the presence of those determinants. What is lacking is a systematic approach for doing so.
The present invention is directed to overcoming the above-identified deficiencies in the art.